The adeno-associated virus (AAV) is a replication-defective parvovirus that has many attractive properties as a potential gene transfer vector for human gene therapy. The difficulty in producing recombinant AAV in high titer is a major obstacle to the investigation of the molecular events involved in AAV infection, and site-specific integration, as well as to the animal trials that are necessary prior to safe consideration of AAV as a gene transfer vector for humans. The current method of producing recombinant AAV (rAAV) involves co-transfection of cells with plasmid DNA encoding the replication and encapsidation proteins of AAV, along =with a separate plasmid encoding the gene of interest flanked by the AAV terminal repeat structures. In the presence of adenovirus (Ad) infection to supply helper functions for replication, rAAV encoding the gene of interest is packaged. The applicant has created cell lines with the AAV rep and cap genes stably integrated. The specific aims of the proposed research are to attempt to increase the titers of rAAV produced by the packaging cell line via several separate strategies: 1) Upregulation of Rep/Cap protein expression by transfecting into the cell lie an additional rep/cap expression cassette, the copy number of which will be amplified via additional selection pressures; 2) Elimination of transfection, integrating the rAAV vector into the rep/cap cell line on a non-homologous plasmid, such that adenovirus infection alone will result in rAAV production; 3) Alternatively, a transfectable element coding all of the adenovirus helper functions will be created and transfected with the rAAV vector; no live adenovirus will contaminate the rAAV produced, and obligate losses of rAAV during Ad purification will be avoided.